Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

• the present invention relates to novel benzotriazoles, to intermediates for their preparation, to pharmaceutical compositions containing them and to their medicinal use.
• the compounds of the present invention are potent inhibitors of MAP kinases, preferably p38 kinase. They are useful in the treatment of inflammation, osteoarthritis, rhematoid arthritis, cancer, reperfusion or ischemia in stroke or heart attack, autoimmune diseases and other disorders.
• MAPKAP kinase-2 and MAPKAP kinase-3 have been identified as downstream substrates of CSBP/p38 which in turn phosphorylate heat shock protein Hsp 27. It is now known that MAPKAP-2 is essential for LPS induced TNF ⁇ biosynthesis [Kotlyarov et al. Nature Cell Biol., 1 , 94 (1999), see also Cohen, P. Trends Cell Biol., 353-361(1997)].
• IL-1 lnterleukin-1
• TNF Tumor Necrosis Factor
• allograft rejections fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, or ARC (AIDS related complex), keloid information, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
• AIDS cachexia secondary to infection or malignancy
• AIDS cachexia secondary to acquired immune deficiency syndrome
• AIDS AIDS
• ARC AIDS related complex
• keloid information scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
• CSBP/p38/RK kinase inhibitors are well known to those skilled in the art.
• International Patent Publication WO 00/40243 published July 13, 2000, refers to pyridine substituted pyridine compounds and states that these compounds are p38 inhibitors.
• International Patent Publication WO 00/63204 published October 26, 2000, refers to substituted azole compounds and states that these compounds are p38 inhibitors.
• International Patent Publication WO 00/31065 published June 2, 2000, refers to certain heterocyclic compounds and states that these compounds are p38 inhibitors.
• International Patent Publication WO 00/06563, published February 10, 2000 refers to substituted imidazole compounds and states that these compounds are p38 inhibitors.
• the present invention relates to a compound of the formula wherein
• the compounds, salts and prodrugs of the present invention can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present invention. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present invention includes all tautomers of the present compounds.
• the present invention also relates to a compound of the formula wherein
• alkyl as well as the alkyl moieties of other groups referred to herein (e.g., alkoxy), may be linear or branched (such as methyl, ethyl, n -propyl, iso propyl, n-butyl, iso -butyl, secondary -butyl, tertiary -butyl); optionally substituted by 1 to 3 suitable substituents as defined above such as fluoro, chloro, trifluoromethyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryloxy, trifluoromethoxy, difluoromethoxy or (C 1 -C 6 )alkyl.
• suitable substituents as defined above such as fluoro, chloro, trifluoromethyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryloxy, trifluoromethoxy, difluoromethoxy or (C 1 -C 6 )al
• cycloalkyl refers to a mono or bicyclic carbocyclic ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl, cyclohexenyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl and bicyclo[5.2.0]nonanyl, etc.); optionally containing 1 or 2 double bonds and optionally substituted by 1 to 3 suitable substituents as defined above such as fluoro, chloro, trifluoromethyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryloxy, trifluoromethoxy, difluoromethoxy or (C 1 -C 6 )alkyl.
• suitable substituents as defined above such as fluoro, chloro, trifluoromethyl,
• (C 2 -C 6 )alkynyl is used herein to mean straight or branched hydrocarbon chain radicals having one triple bond including, but not limited to, ethynyl, propynyl, butynyl, and the like; optionally substituted by 1 to 3 suitable substituents as defined above such as fluoro, chloro, trifluoromethyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryloxy, trifluoromethoxy, difluoromethoxy or (C 1 -C 6 )alkyl.
• heteroaryl refers to an aromatic heterocyclic group usually with one heteroatom selected from O, S and N in the ring.
• the aromatic group may optionally have up to four N atoms in the ring.
• heteroaryl group includes pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl, furyl, imidazolyl, pyrrolyl, oxazolyl (e.g., 1,3-oxazolyl, 1,2-oxazolyl), thiazolyl (e.g., 1,2-thiazolyl, 1,3-thiazolyl), pyrazolyl, tetrazolyl, triazolyl (e.g., 1,2,3-triazolyl, 1,2,4-triazolyl), oxadiazolyl (e.g., 1,2,3-oxadiazolyl), thiadiazolyl (e.g., 1,3,4-thiadiazolyl), quinolyl, isoquinolyl, benzothienyl, benzofuryl, indolyl, and the like; optionally substituted by 1 to 3 suitable substituents as defined above such as fluor
• heteroaryl groups include oxazolyl, imidazolyl, pyridyl, thienyl, furyl, thiazolyl and pyrazolyl (these heteroaryls are most preferred of the R 4 , R 5 , R 6 and R 7 heteroaryls).
• heterocyclic refers to a cyclic group containing 1-9 carbon atoms and 1 to 4 hetero atoms selected from N, O, S or NR'.
• examples of such rings include azetidinyl, tetrahydrofuranyl, imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, thiomorpholinyl, tetrahydrothiazinyl, tetrahydro-thiadiazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl, oxazinyl, oxaithiazinyl, indolinyl, isoindolinyl, quincuclidinyl, chromanyl, isochromanyl, benzoxazinyl, and the like.
• R 2 is optionally substituted (C 3 -C 6 )cycloalkyl, more preferably optionally substituted cyclobutyl or cyclopentyl; more preferably optionally substituted by 1 to 3 substituents selected from the group consiting of fluoro, chloro, trifluoromethyl, hydroxy, (C 1 -C 6 )alkoxy, amino, trifluoromethoxy, and (C 1 -C 6 )alkyl.
• R 2 is optionally substituted (C 1 -C 10 )heteroaryl, more preferably optionally substituted thiophenyl and pyridinyl, more preferably optionally substituted by 1 to 2 substituents independently selected from the group consiting of fluoro, chloro, trifluoromethyl, hydroxy, (C 1 -C 6 )alkoxy, amino, trifluoromethoxy, and (C 1 -C 6 )alkyl.
• phenyl-pyrazolyl-benzotriazoles are those group of compounds of formula I wherein the compound has the formula
• Other embodiments of the present invention include those compounds of formula I(f) in combination with each of the aforementioned embodiments of R 2 .
• Another preferred embodiment of the present invention is that group of compounds of formula I (and I(a), I(c), I(e), I(f), and I(h)) wherein R 4 is hydrogen.
• Other embodiments of the present invention include those compounds of formula I (and I(a), I(c), I(e), I(f), and I(h)) wherein R 4 is hydrogen in combination with the aforementioned embodiments of R 2 .
• Another preferred embodiment of the present invention is that group of compounds of formula I (and I(a), I(c), I(e), I(f), and I(h)) wherein R 4 is R 9 B-(CH 2 ) n -; n is zero; B is a bond and R 9 is R 13 -(R 12 CH) m -.
• Other embodiments of the present invention include those compounds of formula I (and I(a), I(c), I(e), I(f), and I(h)) wherein R 4 is R 9 -B-(CH 2 ) n -, n is zero and R 9 is R 13 -(R 12 CH) m - in combination with the aforementioned embodiments of R 2 .
• R 4 is R 9 -B-(CH 2 ) n -; n is zero; B is -(R 10 -N)-; R 9 is hydrogen or R 13 -(R 12 CH) m -; m is 1 to -6; R 10 is hydrogen or methyl; R 12 is hydrogen or methyl; and R 13 is selected from the group consisting of hydrogen, (C 1 -C 6 )alkyl, hydroxy, (C 1 -C 6 )alkoxy, amino, (C 1 -C 6 )alkylamino, [(C 1 -C 6 )alkyl] 2 amino, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, phenyl, (C 1 -C 10 )heteroaryl, (C 1 -C 10 )heter
• R 4 is R 9 -B-(CH 2 ) n -; n is zero; B is a bond, and R 9 is as described above, in combination with the aforementioned embodiments of R 2 .
• R 4 is R 9 -B-(CH 2 ) n -; n is an integer from one to six, more preferably one to five, more preferably one to three; B is a bond, and R 9 is selected from the group consisting of optionally substituted phenyl, (C 1 -C 10 )heterocyclic, (C 1 -C 10 )heteroaryl and (C 3 -C 10 )cycloalkyl; wherein each of the aforesaid R 9 phenyl, (C 1 -C 10 )heteroaryl, (C 1 -C 10 )heterocyclic and (C 3 -C 10 )cycloalkyl substituents may optionally be substituted by one to four moieties independently selected from the group consisting of halo, (C 1 -C 6 )alky
• inventions include those compounds of formula I (and I(c) and l(f)) wherein R 7 is as defined above, in combination with each of the aforementioned I(c) and I(f) R 4 embodiments and with each of the aforementioned R 2 embodiments.
• Another embodiment of the present invention is that group of compounds of formula I (and I(b)) wherein R 5 is hydrogen.
• Other embodiments of the present invention include those compounds of formula I (and I(b)) wherein R 5 is hydrogen, in combination with each of the aforementioned R 2 embodiments.
• R 5 is said R 14 -(CHR 15 ) p -, p is 1 to -6; and R 14 is as defined above, in combination with each of the aforementioned R 2 embodiments.
• R 5 is said R 14 -(CHR 15 ) p -, p is 1 to 6; and R 14 , R 15 and R 16 are as defined above, in combination with each of the aforementioned R 2 embodiments.
• Another embodiment of the present invention is that group of compounds of formula I (and I(a), I(b), I(d), I(g)) wherein R 6 is R 9 -B-(CH 2 ) n - and n is zero.
• Other embodiments of the present invention include those compounds of formula I (and I(a), I(b), I(d), I(g)) wherein R 6 is R 9 -B-(CH 2 ) n - and n is zero, in combination with each of the aforementioned I(a) R 4 embodiments, I(b) R 5 embodiments or with each of the aforementioned R 2 embodiments.
• inventions include those compounds of formula I (and I(a), I(b), I(d), I(g)) wherein R 6 is R 9 -B-(CH 2 ) n - and n is zero; B is a bond and R 9 is as defined above, in combination with each of the aforementioned I(a) R 4 embodiments, I(b) R 5 embodiments or with each of the aforementioned R 2 embodiments.
• R 6 is R 9 -B-(CH 2 ) n - and n is zero;
• R 9 is as defined above, in combination with each of the aforementioned I(a) R 4 embodiments, I(b) R 5 embodiments or with each of the aforementioned R 2 embodiments.
• R 6 is R 9 -B-(CH 2 ) n -; n is zero; B is a bond; R 9 is R 13 -(R 12 CH) m -; m is 1 to 6; R 10 is hydrogen or methyl; R 12 is hydrogen or methyl; and R 13 is selected from the group consisting of hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, phenyl, (C 1 -C 10 )heteroaryl, (C 1 -C 10 )heterocyclic, (C 3 -C 10 )cycloalkyl, amino, (C 1 -C 6 )alkylamino, [(C 1 -C 6 )alkyl] 2 amino, (C 1 -C 6 )alkyl-SO 2 -NH-, phenyl-SO 2
• R 6 is R 9 -B-(CH 2 ) n - and n is zero; B is a bond; R 9 is R 13 -(R 12 CH) m -; m is 1 to 6; R 10 is hydrogen or methyl; R 12 is hydrogen or methyl; and R 13 is as defined above, in combination with each of the aforementioned I(a) R 4 embodiments, I(b) R 5 embodiments or with each of the aforementioned R 2 embodiments.
• R 6 is R 9 -B-(CH 2 ) n -; n is an integer from one to six, more preferably one to five, more preferably one to three; B is a bond, and R 9 is as described above, in combination with each of the aforementioned I(a) R 4 embodiments, I(b) R 5 embodiments or with each of the aforementioned R 2 embodiments..
• R 6 is R 9 -B-(CH 2 ) n -; n is an integer from one to six, more preferably one to five, more preferably one to three; B is a bond, and R 9 is R 13 -(R 12 CH) m -; m is 1 to 6; R 10 is hydrogen or methyl; each R 12 is independently selected from the groups consisting of hydrogen or methyl; and R 13 is selected from the group consisting of hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, phenyl, (C 1 -C 10 )heteroaryl, (C 1 -C 10 )heterocyclic, (C 3 -C 10 )cycloalkyl, hydroxy, (C 1 -C 6 )alkoxy, perhalo(C 1 -C 6 )alkoxy,
• R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• Another embodiment of the present invention is that group of compounds of formula I wherein s is an integer from zero to four and one of R 3 is selected from the group consisting of optionally substituted phenyl, (C 1 -C 10 )heteroaryl, (C 1 -C 10 )heterocyclic and (C 3 -C 10 )cycloalkyl.
• Other embodiments of the present invention include those compounds of formula I (and I(a), I(b), I(c), I(d), I(e), I(f) and I(g)) wherein R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• Another embodiment of the present invention is that group of compounds of formula I wherein s is an integer from zero to four and one of R 3 is selected from the group consisting of hydroxy, (C 1 -C 6 )alkoxy, perhalo(C 1 -C 6 )alkoxy, phenoxy, (C 1 -C 10 )heteroaryl-O-, (C 1 -C 10 )heterocyclic-O-, (C 3 -C 10 )cycloalkyl-O-, (C 1 -C 6 )alkyl-S-, (C 1 -C 6 )alkyl-SO 2 - and (C 1 -C 6 )alkyl-NH-SO 2 -.
• R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• Preferred compounds of the present invention is that group of compounds of formula I wherein s is an integer from zero to two and each R 3 is independently selected from the group consisting of halo, (C 1 -C 6 )alkyl, perhalo(C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, perhalo(C 1 -C 6 )alkoxy and -CN.
• More preferred compounds of the present invention is that group of compounds of formula I wherein s is an integer from zero to three and each R 3 is independently selected from the group consisting of fluoro, chloro and methyl.
• Other embodiments of the present invention include those compounds of formula I (and I(a), I(b), I(c), I(d), I(e), I(f) and I(g)) wherein R 3 is as defined above in combination with each of the aforementioned R 6 embodiments, R 7 embodiments, R 4 embodiments, R 5 embodiments or with each of the aforementioned R 2 embodiments.
• An embodiment of the (isoxazole-5-yl)-benzoimidazoles are those compounds wherein R 4 is hydrogen.
• Another embodiment of the (isoxazole-5-yl)-benzoimidazoles are those compounds wherein R 4 is other than hydrogen.
• this invention relates to a method of inhibiting the production of IL-1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• Excessive or unregulated TNF production has been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, cerebral malaria, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, such as osteoporosis, cardiac, brain and renal reperfusion injury, graft vs.
• diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, cerebral
• viruses of Formula (I) are also useful in the treatment of viral infections, where such viruses are sensitive to upregulation by TNF or will elicit TNF production in vivo .
• the viruses contemplated for treatment herein are those that produce TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirectly, by the TNF inhibiting-compounds of Formula (I).
• viruses include, but are not limited to HIV-1, HIV-2 and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex.
• Abnormal levels of IL-1, IL-6, IL-8 or TNF constitute: (i) levels of free (not cell bound) IL-1, IL-6, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) any cell associated IL-1, IL-6, IL-8 or TNF; or (iii) the presence of IL-1, IL-6, IL-8 or TNF mRNA above basal levels in cells or tissues in which IL-1, IL-6, IL-8 or TNF, respectively, is produced.
• IL-1 IL-6, IL-8 or TNF
• TNF mediated disease or disease state refers to any and all disease states in which TNF plays a role, either by production of TNF itself, or by TNF causing another monokine to be released, such as but not limited to IL- 1, IL-6 or IL-8.
• cytokine biosynthesis inhibitors of the, present invention, compounds of Formula (I) have been detemined to be potent and selective inhibitors of CSBP/p38/RK kinase activity, These inhibitors are of aid in determining the signaling pathways involvement in inflammatory responses.
• a definitive signal transduction pathway can be prescribed to the action of lipopolysaccharide in cytokine production in macrophages.
• treatment of stroke neurotrauma/CNS head injury, cardiac, brain and renal reperfusion injury, thrombosis, glomerulonephritis, diabetes and pancreatic ⁇ cells, multiple sclerosis, muscle degeneration , eczema, psoriasis, sunburn, and conjunctivitis are also included.
• vasculature inappropriate angiogenesis includes, but is not limited to, diseases which are characterized by hemangiomas and ocular diseases.
• the present invention provides a method of treating a p38 kinase mediated disease in a mammal in need thereof, preferably a human, which comprises administering to said mammal, an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• treating refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
• treatment refers to the act of treating, as “treating” is defined immediately above.
• a mammal including a human, comprising an amount of a compound of formula I effective in such treatment and a pharmaceutically acceptable carrier.
• compositions containing prodrugs of compounds of the formula I can be converted into prodrugs.
• Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of compounds of formula I.
• the present invention also encompasses sustained release compositions.
• the compounds of the invention are useful in treating a diverse array of diseases.
• One of ordinary skill in the art will also appreciate that when using the compounds of the invention in the treatment of a specific disease that the compounds of the invention may be combined with various existing therapeutic agents used for that disease.
• the compounds of the invention may be combined with agents such as TNF- ⁇ inhibitors such as anti-TNF monoclonal antibodies (such as Remicade, CDP-870 and D 2 E 7 ) and TNF receptor immunoglobulin molecules (such as Enbrel®), COX-2 inhibitors (such as meloxicam, celecoxib , rofecoxib, valdecoxib and etoricoxib) low dose methotrexate, leflunomide, hydroxychloroquine, d-penicillamine, auranofin or parenteral or oral gold.
• TNF- ⁇ inhibitors such as anti-TNF monoclonal antibodies (such as Remicade, CDP-870 and D 2 E 7 ) and TNF receptor immunoglobulin molecules (such as Enbrel®)
• COX-2 inhibitors such as meloxicam, celecoxib , rofecoxib, valdecoxib and etoricoxib
• methotrexate such as meloxicam
• Suitable agents to be used in combination include standard non-steroidal anti-inflammatory agents (hereinafter NSAID's) such as piroxicam, diclofenac, propionic acids such as naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone, salicylates such as aspirin, COX-2 inhibitors such as celecoxib, valdecoxib, rofecoxib and etoricoxib, analgesics and intraarticular therapies such as corticosteroids and hyaluronic acids such as hyalgan and synvisc.
• NSAID's standard non-steroidal anti-inflammatory agents
• piroxicam such as piroxicam, diclofenac, propionic acids such as naproxen, flubiprof
• the compounds of the present invention may also be used in combination with anticancer agents such as endostatin and angiostatin or cytotoxic drugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, farnesyl transferase inhibitors, VegF inhibitors, and antimetabolites such as methotrexate.
• anticancer agents such as endostatin and angiostatin or cytotoxic drugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, farnesyl transferase inhibitors, VegF inhibitors, and antimetabolites such as methotrexate.
• the compounds of the invention may also be used in combination with antiviral agents such as Viracept, AZT, aciclovir and famciclovir, and antisepsis compounds such as Zovant, tifacogin, NOX-100 and GR270773.
• antiviral agents such as Viracept, AZT, aciclovir and famciclovir
• antisepsis compounds such as Zovant, tifacogin, NOX-100 and GR270773.
• the compounds of the present invention may also be used in combination with cardiovascular agents such as calcium channel blockers, lipid lowering agents such as statins, fibrates, beta-blockers, Ace inhibitors, Angiotensin-2 receptor antagonists and platelet aggregation inhibitors.
• cardiovascular agents such as calcium channel blockers, lipid lowering agents such as statins, fibrates, beta-blockers, Ace inhibitors, Angiotensin-2 receptor antagonists and platelet aggregation inhibitors.
• the compounds of the present invention may also be used in combination with CNS agents such as antidepressants (such as sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists, Nicotine agonists, Dopamine agonists and inhibitors of neuronal nitric oxide synthase), and anti-Alzheimer's drugs such as donepezil, tacrine, COX-2 inhibitors, propentofylline or metryfonate.
• CNS agents such as antidepressants (such as sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors,
• the compounds of the present invention may also be used in combination with osteoporosis agents such as roloxifene, droloxifene, lasofoxifene or fosomax and immunosuppressant agents such as FK-506 and rapamycin.
• osteoporosis agents such as roloxifene, droloxifene, lasofoxifene or fosomax
• immunosuppressant agents such as FK-506 and rapamycin.
• Scheme 1 refers to the preparation of compounds of the formula I.
• a compound of the formula I wherein (R 3 ) s -phenyl-Het is (c) or (f) can be prepared from compounds of the formula II by reaction with an aminating reagent.
• Suitable aminating reagents include hydrazines of the formula H 2 N-NH-R 7 , in a polar solvent.
• Suitable solvents include alcohols such as ethanol, propanol or butanol or mixtures of alcohols and acetic acid, preferably ethanol.
• the aforesaid reaction is conducted at a temperature of about 10°C to about 30°C, preferably at about 22°C, for a period from about 1 hour to about 7 hours, preferably about 3 hours.
• the compound of formula II is prepared from a compound of formula VI by reaction with an acetal, such as dimethylformamide - dimethylacetal, at a temperature of about 60°C to about 90°C, preferably about 80°C for a period from about 1 hour to about 6 hours, preferably about 3 hours.
• an acetal such as dimethylformamide - dimethylacetal
• compounds of the formula I wherein (R 3 ) s -phenyl-Het is (c) or (f), can be prepared from compounds of the formula III by reaction with an aminating reagent such as H 2 N-NH-R 7 according to methods analogous to the conversion of compounds of formula II to formula I, above.
• the compound of formula III is prepared from a compound of formula VI by reaction with an isothiocyanate.
• Suitable solvents for the aforesaid reaction include pyridine, N,N-dimethylformamide or tetrahydrofuran, preferably pyridine. The aforesaid reaction is performed from a period of about 0.5 hour to about 4 hours at a temperature of about 0°C to about 30°C.
• the compound of formula IV is prepared from a compound of formula V by reaction with sodium methoxide, or sodium ethoxide, or sodium tert-butoxide, preferably sodium methoxide, in an alcohol solvent, such as methanol, ethanol, isopropanol, preferably methanol.
• the aforesaid reaction can be conducted at a temperature of about 0°C to about 30°C, preferably at about 22°C, for a period of time from 15 minutes to about 3 hours, preferably about 30 minutes.
• the aforesaid reaction is followed by an aqueous acidic work-up.
• the compound of formula V is prepared from a compound of formula VI by reaction with bromine (Br 2 ) in a polar solvent.
• Suitable solvents include acetic acid, chloroform or methylene chloride, preferably acetic acid.
• the aforesaid reaction is conducted at a temperature of about 0°C to about 30°C preferably at about 22°C (room temperature) for a period from about 10 minutes to about 4 hours, preferably about 30 minutes.
• Compounds of the formula I, wherein (R 3 ) s -phenyl-Het is (a), can be prepared from compounds of the formula VII, by reaction with an ammonia source and cuprous acetate and a polar solvent.
• Suitable ammonia sources include ammonium trifluoroacetate, ammonia, and ammonium acetate, preferably ammonium acetate.
• the aforesaid reaction can be run neat or in the presence of a solvent such as alcohols (methanol, ethanol or butanol) and acetic acid.
• the aforesaid reaction can be run at a temperature from about 20°C to about 80°C for a period from about 15 minutes to about 4 hours, preferably neat conditions at about 60°C for about 2 hours.
• compounds of formula I (g) and (h) can be prepared from compounds of formula VI according to methods described in the literature (Gauthier, J. Y.; Leblanc, Y.; Black, C.; Chan, C.-C.; Cromlish, W. A.; Gordon, R.; Kennedey, B. P.; Lau, C. K.; Léger, S.; Wang, Z.; Ethier, D.; Guay, J.; Mancini, J.; Riendeau, D.; Tagari, P.; Vickers, P.; Wong, E.; Xu, L.; Prasit, P. Bioorg. Med. Chem. Lett. 1996, 6, 87-92).
• the compound of formula VII is prepared from a compound of formula VI by reaction with a reagent of the formula wherein L is a leaving group such as chloro, bromo, iodo or mesylate, in the presence of a base and a solvent.
• Suitable bases include sodium hydride (NaH) and n-butyllithium.
• Suitable solvents include tetrahydrofuran (THF) and dimethyl formamide (DMF).
• the aforesaid reaction can be conducted at a temperature from about -30°C to about the reflux temperature of the solvent, for a period of about 5 minutes to about 24 hours.
• the compound of formula VI is prepared according to the methods. of Davies, I. W.; Marcoux, J.-F.; Corley, E. G.; Journet, M.; Cai, D.-W.; Palucki, M.; Wu, J.; Larsen, R. D.; Rossen, K.; Pye, P. J.; DiMichele, L.; Dormer, P.; Reider, P. J.; J. Org. Chem. , Vol. 65, pp. 8415-8420 (2000).
• the compound of formula VIII is prepared by methods well known to those skilled in the art.
• a compound of the formula I wherein (R 3 ) s -phenyl-Het is a group of the formula (b), can be prepared from a compound of the formula X by reaction with a compound of the formula wherein R 5 is hydrogen, in the presence of a polar solvent.
• Suitable solvents include dimethyl formamide, chloroform, DMSO, THF and ethanol, preferably dimethylformamide.
• the aforesaid reaction is conducted at a temperature of about 15°C to about 80°C, preferably about 60°C, for a period from about 4 hours to about 4 days, preferably 4 hours.
• Suitable ammonia sources include ammonium trifluoroacetate, ammonia, and ammonium acetate, preferably ammonium acetate.
• the aforesaid reaction can be run neat or in the presence of a solvent such as alcohols (methanol, ethanol or butanol) and acetic acid.
• the aforesaid reaction can be run at a temperature from about 20°C to about 80°C for a period from about 15 minutes to about 4 hours, preferably neat conditions at about 60°C for about 2 hours.
• the compound of formula XII is prepared from a compound of the formula XIII by reaction with an oxidizing reagent in a polar protic solvent.
• Suitable oxidizing reagents include copper acetate, pyridiniumchlorochromate (PCC) and tetrapropylammonium peruthenate/N-methyl morpholine-N-oxide (TPAP/NMO), preferably cuprous acetate.
• Suitable solvents include acetic acid.
• the aforesaid reaction can be run neat or in the presence of a solvent such as alcohols (methanol, ethanol or butanol) and acetic acid.
• the aforesaid reaction can be run at a temperature from about 20°C to about 80°C for a period from about 15 minutes to about 4 hours, preferably neat conditions at about 60°C for about 2 hours.
• the compound of formula XIII is prepared from a compound of the formula X by reaction with a methoxide such as described in Scheme 1 for the preparation of compounds of formula IV from compounds of formula V.
• the compound of formula X is prepared from a compound of the formula XIV by reaction with Br 2 in a polar solvent.
• Suitable solvents include acetic acid, chloroform or methylene chloride, preferably acetic acid.
• the aforesaid reaction is conducted at a temperature of about 0°C to about 30°C preferably at about 22°C (room temperature) for a period from about 10 minutes to about 4 hours, preferably about 30 minutes.
• Scheme 3 refers to an alternate preparation of compounds of formula I, wherein (R 3 ) s -phenyl-Het is a group of the formula (b) or (d) and R 6 is hydrogen.
• a compound of formula I wherein (R 3 ) s -phenyl-Het is of the formula (d) and R 6 is hydrogen, is prepared from a compound of formula XVII by reaction with an isocyanide of formula in the presence of a base.
• Suitable bases include potassium carbonate, triethylamine, and piperazine, preferably potassium carbonate.
• Suitable solvents include polar solvents such as tetrahydrofuran, or N,N-dimethylformamide, preferably in N,N-dimethylformamide.
• the aforesaid reaction may be run at a temperature between about 22°C and about 70°C, preferably at about 22°C for a period from about 2 hours to about 4 hours, followed by about 6 hours to about 10 hours at a temperature of about 70°C.
• the imine of formula XV can be prepared and subsequently reacted in an aqueous media as described in the literature: (Sisko, J.; Kassik, A. J.; Mellinger, M.; Filan, J. J.; Allen, A.; Olsen, M. A.; J. Org. Chem. , 65, 1516-1524 (2000)). Reactions of imines of formula XV with suitable isocynanides of formula XVI are conducted at about 22°C for a time period from about 1 day to about 21 days, preferably about 1 day.
• a compound of formula XVII is prepared from a compound of formula XXIII in Scheme 4.
• the compound of formula XVIII is prepared form a compound of formula XIX by reaction with an oxidizing reagent such as N-methyl morpholine N-oxide/ TPAP, Dess-Martin reagent, PCC or oxalyl chloride-DMSO , preferably N-methyl morpholine N-oxide/ TPAP.
• an oxidizing reagent such as N-methyl morpholine N-oxide/ TPAP, Dess-Martin reagent, PCC or oxalyl chloride-DMSO , preferably N-methyl morpholine N-oxide/ TPAP.
• Suitable solvents for the aforesaid reaction include methylene chloride, chloroform, THF or dichloromethane.
• the aforesaid reaction is conducted at a temperature from about 10°C to about 30°C for a time from about 15 minutes to about 3 hours, preferably about 1 hour.
• the compound of formula XIX is prepared from a compound of the formula XX by reaction with an acylating reagent of the formula, wherein L is a leaving group, and a base.
• Suitable bases include triethylamine, Hunig's base, or DBU, preferably triethylamine.
• Suitable leaving groups include Cl, Br or activated acids.
• Suitable solvents for the aforesaid reaction include methylene chloride, dimethyl formamide, THF or DMF, and mixtures thereof, preferably methylene chloride.
• the aforesaid reaction is conducted at a temperature from about 10°C to about 30°C preferably about 22°C (room temperature) for a period from about 1 hour to about 6 hours preferably about 1 hour.
• the compound of the formula XX is prepared form a compound of formula XXII by reaction with a reducing agent.
• Reducing agents are well known to those skilled in the art.
• reduction of the double bond may be effected with hydrogen gas (H 2 ), using catalysts such as palladium on carbon (Pd/C), palladium on barium sulfate (Pd/BaSO 4 ), platinum on carbon (Pt/C), or tris(triphenylphosphine) rhodium chloride (Wilkinson's catalyst), in an appropriate solvent such as methanol, ethanol, THF, dioxane or ethyl acetate, at a pressure from about 1 to about 5 atmospheres and a temperature from about 10°C to about 60°C, as described in Catalytic Hydrogenation in Organic Synthesis , Paul Rylander, Academic Press Inc., San Diego, 31-63 (1979).
• Pd on carbon methanol at 25°C and 50 psi
• the compound of the formula XIV is prepared from a compound of the formula XXIII by reaction with a Grignard reagent of the formula (R 3 ) s -phenyl-CH 2 -M, wherein M is magnesium chloride or magnesium bromide.
• Suitable solvents for the aforesaid reaction are ethers (such as dimethyl ether THF, DME or dioxane), preferably THF.
• the aforesaid reaction is conducted at a temperature from about 0°C to about 30°C, preferably at about 22°C (room temperature), for a period from about 1 hour to about 48 hours, preferably about 6 hours.
• Scheme 5 refers to the preparation of compounds of the formula I, wherein (R 3 ) s -phenyl-Het is a group of the formula (e).
• a compound of the formula I can be prepared from compound of formula XXIV by reaction with a hydroxylamine (preferably a salt thereof such as the hydrochloride salt), and a base.
• Suitable bases include pyridine or a trialkylamine, preferably pyridine.
• Suitable solvents include N,N-dimethylformamide, tetrahydrofuran or pyridine, preferably pyridine.
• the aforesaid reaction is conducted at a temperature from about 0°C to about 100°C, preferably at about 60°C, for a period from about 1 hour to about 48 hours, preferably about 20 hours.
• the compound of formula XXIV can be prepared from a compound of formula XXV by reaction with an ester of the formula (R 3 ) s -phenyl-CO 2 P 1 , wherein P 1 is methyl or ethyl, in the presence of a base and a solvent.
• Suitable bases include sodium hydride, lithium diisopropylamide, or sodium alkoxides, preferably sodium ethoxide.
• Suitable solvents include alcohols such as methanol, ethanol, propanol, butanol, or tetrahydrofuran, preferably ethanol.
• the aforesaid reaction is conducted at a temperature from about 23°C to about 65°C, preferably at about 50°C, for a period from about 2 hours to about 24 hours, preferably about 20 hours.
• the compound of formula XXV can be made by methods well known to those of ordinary skill in the art and in Scheme 7 from compounds of formula XXXI by reaction with a halogenating reagant followed by reaction with a nitrile according to methods well known to those of ordinary skil in the art.
• Scheme 6 refers to the preparation of compounds of the formula XVII and XXIII which are intermediates for the preparation of compounds of formula I in Schemes 3 and 4, respectfully.
• a compound of the formula XVII is prepared from a compound of formula XXIII by reaction with a reducing agent, such as diisobutylaluminum hydride (DiBAl) in toluene, in a solvent, such as tetrahydrofuran (THF).
• a reducing agent such as diisobutylaluminum hydride (DiBAl) in toluene
• a solvent such as tetrahydrofuran (THF).
• the foresaid reaction may be run at a temperature from about -78°C to room temperature for a period from about one to about five hours.
• the compound of formula XXIII is prepared from a compound of formula XXVI by reaction with a suitable activating agent and a compound of the formula, and a base.
• Suitable activating agents include thionyl chloride, EDCI and DCC, preferably oxalyl chloride.
• Suitable bases include triethylamine, Hunig's base, or DBU, preferably triethylamine.
• Suitable solvents for the aforesaid reaction include methylene chloride, dimethyl formamide, THF or DMF, and mixture thereof, preferably methylene chloride.
• the aforesaid reaction is conducted at a temperature from about 0°C to about 30°C preferably about 22°C (room temperature) for a period from about 6 hours to about 48 hours preferably about 12 hours.
• the compound of formula XXVI is prepared from a compound of formula XXVIII by reaction with sodium nitrite under acidic conditions. Suitable acids include hydrochloric acid. The aforesaid reaction is conducted at a temperature from about 0°C to about 100°C, preferably about 22°C, for a period from about 1 hour to about 3 hours, preferably about 2 hours.
• the compound of formula XXVIII is prepared from a compound of formula XXIX by reaction with a reducing agent.
• Reducing agents are well known to those skilled in the art.
• reduction of the double bond may be effected with hydrogen gas (H 2 ), using catalysts such as palladium on carbon (Pd/C), palladium on barium sulfate (Pd/BaSO 4 ), platinum on carbon (Pt/C), or tris(triphenylphosphine) rhodium chloride (Wilkinson's catalyst), in an appropriate solvent such as methanol, ethanol, THF, dioxane or ethyl acetate, at a pressure from about 1 to about 5 atmospheres and a temperature from about 10°C to about 60°C, as described in Catalytic Hydrogenation in Organic Synthesis. Paul Rylander, Academic Press Inc., San Diego, 31-63 (1979). The following conditions are preferred: Pd on carbon, methanol at 25°C and 50 p
• the compound of formula XXIX can be prepared from a compound of formula XXX by reaction with an amine of the formula R 2 NH 2 .
• Suitable solvents include an excess of the amine reactant (neat), glyme, and toluene, preferably neat.
• the aforesaid reaction is conducted at a temperature from about 70°C to about 120°C, preferably 100°C, for a period from about 10 minutes to about 1 hour, preferably about 30 minutes.
• Scheme 7 refers to an alternate preparation of compounds of the formula XVII which are intermediates useful in the preparation of compounds of formula I in Scheme 3.
• a compound of the formula XVII is prepared from a compound of formula XXXI by reaction with an oxidizing reagent such as N-methyl morpholine N-oxide/ TPAP, Dess-Martin reagent, PCC or oxalyl chloride-DMSO, preferably oxalyl chloride-DMSO.
• Suitable solvents for the aforesaid reaction include methylene chloride, chloroform, THF or dichloromethane.
• the aforesaid reaction is conducted at a temperature from about -78°C to about 22°C for a time from about 15 minutes to about 3 hours, preferably about 1 hour.
• the compound of the formula XXXI is prepared from a compound of the formula XXXII by reaction with a reducing reagant.
• Suitable reducing agents include lithium borohydride, sodium borohydride (NaBH 4 ), sodium cyanoborohydride (NaCNBH 3 ), lithium aluminum hydride (LiAIH 4 ) and borane in THF (BH 3 ⁇ THF).
• Suitable solvents include methanol, ethanol, THF, diethyl ether, dioxane and tetrahydrofuran.
• the aforesaid reaction is conducted at a temperature from about 0°C to about 70°C, preferably 65°C, for a period from about 10 minutes to about 1 hour, preferably about 30 minutes.
• Scheme 8 refers to an alternate preparation of compounds of the formula XXVI, wherein R 2 is optionally substituted (C 1 -C 6 )alkyl, (C 3 -C 10 )cycloalkyl, and (C 1 -C 10 )heterocyclic; which are intermediates in Scheme 6 useful in the preparation of compounds of formula I, wherein R 2 is optionally substituted (C 1 -C 6 )alkyl, (C 3 -C 10 )cycloalkyl, and (C 1 -C 10 )heterocyclic.
• a compoound of formula XXVI is prepared from a compound of formula XXVIII by reaction with sodium nitrite under acidic conditions. Suitable acids include hydrochloric acid. The aforesaid reaction is conducted at a temperature from about 0°C to about 100°C, preferably about 22°C, for a period from about 1 hour to about 3 hours, preferably about 2 hours.
• Suitable reducing agents include sodium borohydride, sodium cyanoborohydride and sodium triacetoxyborohydride, preferably triacetoxyborohydride.
• Suitable solvents include acetic acid, THF, DMF and dimethylsulfoxide, prefereably a mixture of acetic acid, THF and DMF. The aforesaid reaction is conducted at a temperature from about 0°C to about 30°C, preferably about 22°C, for a period from about 10 minutes to about 2 hours, preferably about 1 hour.
• the compounds of the formula I which are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to animals, it is often desirable in practice to initially isolate a compound of the formula I from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent, and subsequently convert the free base to a pharmaceutically acceptable acid addition salt.
• the acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is obtained.
• non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium, calcium and magnesium, etc.
• These salts can easily be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, preferably under reduced pressure.
• they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before.
• stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum product yields.
• Mononuclear cells are isolated from heparinized blood (1.5 ml of 1000 units / ml heparin for injection, Elkins-Sinn,lnc. added to each 50 ml sample) using Accuspin System-Histopaque-1077 tubes® (Sigma A- 7054). Thirty-five milliliters of whole blood are added to each tube and the tubes are centrifuged at 2100 rpm for 20 minutes in a Beckman GS-6KR centrifuge with the brake off at room temperature.
• the mononuclear cells which collect at the interface are removed, diluted with Macrophage serum free medium (Gibco-BRL) (Medium) to achieve a final volume of 50 ml, and collected by centrifugation for 10 minutes. The supernatant is discarded and the cell pellet is washed 2 times with 50 ml of Medium. A sample of the suspended cells is taken before the second wash for counting. Based on this count, the washed cells are diluted with Medium containing 1% FBS to a final concentration of 2.7 X 10 6 cells / ml and 75 ⁇ l of the cell suspension is added to each well of a 96 well plate.
• Macrophage serum free medium Gibrophage serum free medium
• Medium Medium containing 1% FBS
• Serial dilutions (1/5) are performed by transferring 20 ⁇ l of this dilution to 80 ⁇ l of Medium containing both LPS and 0.4% DMSO, resulting in solutions containing 8 ⁇ M, 1.6 ⁇ M, 0.32 ⁇ M and 0.064 ⁇ M of test agent.
• the assay is initiated by adding 25 ⁇ l of the diluted compounds to the mononuclear cell suspension and incubating the cells at 37 C and 5% CO 2 for 4 hours.
• the 96-well plates are then centrifuged for 10 minutes at 2000 rpm at 4°C in a Beckman GS-6KR centrifuge to remove cells and cell debris. A 90 ⁇ l aliquot of each supernatant is removed and transferred to a 96 well round bottom plate, and this plate is centrifuged a second time to insure that all cell debris is removed. 80 ⁇ l of the supernatant is removed and transferred to a new round bottom plate.
• Supernatants are analyzed for TNF- ⁇ content using R&D ELISA. 25 ⁇ l of each sample is added to an ELISA well containing 25 ⁇ l of assay diluent RD1F and 75 ⁇ l of assay diluent RD5. The assay is run following kit directions except 100 ⁇ l of conjugate and substrate solutions are used.
• An individual monocyte cell extract (prepared above) is then transferred to each tube containing a pellet of IgG-coated Protein G-Sepharose, and these mixtures are incubated for 2 hours at 4°C (with rocking).
• the beads subsequently are harvested by centrifugation, and the resulting bead pellets are washed once with 0.5 ml of Buffer A containing 0.5 M NaCI, once with 0.5 ml of Buffer A, and once with 0.1 ml of a buffer composed of 20 mM MOPS, pH 7.2, 25 mM sodium ⁇ -glycerophosphate 5 mM EGTA, 1 mM orthovanadate, and 1 mM dithiothreitol (Buffer B).
• a kinase reaction mixture stock is prepared as follows: 2.2 ⁇ l of 10 mCi/ml ⁇ [ 32 P]ATP, 88 ⁇ l of 1.3 ⁇ g/ml solution of MAPKAP Kinase-2 substrate peptide (Upstate Biotechnology #12-240), 11 ⁇ l of 10 mM ATP, 8.8 ⁇ l of 1 M MgCl 2 , and 770 ⁇ l of Buffer B. To each of the immune complex-Protein G-pellets, 40 ⁇ l of the kinase reaction mixture are added and the tubes are incubated for 30 minutes at 30°C.
• the tubes then are clarified by centrifugation and 25 ⁇ l of each supernatant is spotted onto a P81 filter paper disk (Whatman #3698-023). After allowing all fluid to soak into the filter, each disk is placed into an individual well of 6-well cluster plates and the filters are washed sequentially with 2 ml of 0.75% phosphoric acid (3 washes/15 min each) and once with acetone (10 min). The filters then are air dried and transferred to liquid scintillation vials containing 5 ml of scintillation fluid. Radioactivity is determined in a liquid scintillation counter. The amount of radioactivity bound to the filter at each test agent concentration is expressed as a percentage of that observed from cells stimulated with LPS in the absence of a test agent.
• Rats were weighed and dosed with vehicle (0.5% methyl cellulose, Sigma) or drug. One hour later, animals were injected i.p. with LPS (50 ug/rat, Sigma L-4130). Ninety minutes later, animals were sacrificed by asphyxiation with CO 2 and bled by cardiac puncture. Blood was collected in Vaccutainer tubes and spun for 20 minutes at 3000 rpm. Serum was assayed for TNF ⁇ levels using an ELISA (R&D Systems).
• the composition may take the form of tablets or lozenges formulated in conventional manner.
• the compounds of formula I can also be formulated for sustained delivery according to methods well known to those of ordinary skill in the art. Examples of such formulations can be found in United States Patents 3,538,214, 4,060,598, 4,173,626, 3,119,742, and 3,492,397, which are herein incorporated by reference in their entirety.
• the active compounds of the invention may be formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• the active compounds of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
• a proposed dose of the active compounds of the invention for oral, parenteral or buccal administration to the average adult human for the treatment of the conditions referred to above (inflammation) is 0.1 to 200 mg of the active ingredient per unit dose which could be administered, for example, 1 to 4 times per day.
• Aerosol formulations for treatment of the conditions referred to above in the average adult human are preferably arranged so that each metered dose or "puff' of aerosol contains 20 ⁇ g to 1000 ⁇ g of the compound of the invention.
• the overall daily dose with an aerosol will be within the range 100 ⁇ g to 10 mg.
• Administration may be several times daily, for example 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses each time.
• Aerosol combination formulations for treatment of the conditions referred to above are preferably arranged so that each metered dose or "puff" of aerosol contains from about 1 ⁇ g to 1000 ⁇ g of the compound of the invention.
• the overall daily dose with an aerosol will be within the range 100 ⁇ g to 10 mg.
• Administration may be several times daily, for example 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses each time.
• Aerosol formulations for treatment of the conditions referred to above are preferably arranged so that each metered dose or "puff" of aerosol contains from about 20 ⁇ g to 1000 ⁇ g of the compound of the invention.
• the overall daily dose with an aerosol will be within the range 100 ⁇ g to 10 mg of the p38 kinase inhibitor.
• Administration may be several times daily, for example 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses each time.
• compositions containing and methods of treating or preventing comprising administering prodrugs of compounds of the formula I.
• Compounds of formula I having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs.
• Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of compounds of formula I.
• the amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, omithine and methionine sulfone.
• Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of formula I through the carbonyl carbon prodrug sidechain.
• the mass spectrum of the major eluting component was then obtained in positive or negative ion mode scanning a molecular weight range from 165 AMU to 1100 AMU. Specific rotations were measured at room temperature using the sodium D line (589 nm). Commercial reagents were utilized without further purification.
• THF refers to tetrahydrofuran.
• DMF refers to N,N-dimethylformamide.
• Chromatography refers to column chromatography performed using 32-63 mm silica gel and executed under nitrogen pressure (flash chromatography) conditions. Room or ambient temperature refers to 20-25°C. All non-aqueous reactions were run under a nitrogen atmosphere for convenience and to maximize yields. Concentration at reduced pressure means that a rotary evaporator was used.
• 3-Ethyl-3H-benzotriazole-5-carbonitrile (0.065g) was diluted with lithium hydroxide (2 M in water) then heated to 100°C for 2 hours. The mixture was then cooled to 23 C, acidified with 1 N hydrochloric acid and extracted with ethyl acetate. The organic layer was dried with sodium sulfate and concentrated to give 3-ethyl-3H-benzotriazole-5-carboxylic acid (0.059 g).
• 3-Ethyl-3H-benzotriazole-5-carboxylic acid (0.15 g) was diluted with methanol and treated with HCI (gas). The reaction was then heated to 65°C for 18 hours. The mixture was allowed to cool to 23°C and was diluted with saturated sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried with sodium sulfate and concentrated to give 3-ethyl-3H-benzotriazole-5-carboxylic acid methyl ester (0.143 g).
• 3-Ethyl-3H-benzotriazole-5-carboxylic acid methyl ester (0.143 g) was diluted with tetrahydrofuran (2 mL) and methanol (0.06 mL). Lithium borohydride (0.023 g) was added and the solution was allowed to stand at 23°C for 30 minutes. The reaction was then poured onto saturated ammonium chloride and extracted with ethyl acetate. The organic layer was dried with sodium sulfate and concentrated to give (3-Ethyl-3H-benzotriazol-5-yl)-methanol (0.13 g).
• 3-Ethyl-3H-benzotriazole-5-carbaldehyde was prepared from (3-Ethyl-3H-benzotriazol-5-yl)-methanol by a Swern oxidation.

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